B. burgdorferi is maintained in nature in two very distinct environments, the Ixodes tick and mammalian hosts. Its ability to adapt and survive in these very different environments is attributed to its ability to sense changes in temperature, pH, cell density, oxygen and/or exposure to host factors and alter gene expression accordingly. The capability of bacteria to regulate transcription initiation allows for the coordinated expression of genes at appropriate times. Previous reports have demonstrated that central to the regulation of these responses are SigmaS (encoded by rpoS) and Sigma54 (encoded by rpoN also known as ntrA). In addition, Sigma54-dependent expression of SigmaS is responsible for the expression of key virulence factors e.g., outer surface protein C (OspC), OspA and decorin-binding protein C (DbpC) required for infectivity and transmission during the infective cycle. Promoters recognized by Sigma54-RNA polymerase holoenzyme (Sigma54-holoenzyme) have well conserved GG and GC doublets at Sigma4 and Sigma12 positions, respectively, relative to the transcriptional start site, instead of the typical -35/-10 boxes observed in Sigma70 class promoters. Universally, Sigma54-dependent transcription has been shown to require activator proteins, many of which are response regulators of two-component systems. In most cases, Sigma54-activator proteins bind enhancer-like elements located 100-200 bp upstream of the transcriptional start site of the promoter. Analysis of the genome of B. burgdorferi identified Rrp2 (BBO763) as a predicted Sigma54-dependent activator consisting of an N-terminal receiver domain, a central ATPase domain and a C-terminal DNA-binding domain. The activities of activators of Sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of #m54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. An activator of Sigma54-holoenzyme in B. burgdorferi, Rrp2 (encoded by rrp2), is also a response regulator of a two-component system and rrp2 is in an operon with a gene encoding its cognate protein histidine kinase, hk2 (encoded by hk2). The Sigma54-dependent activation of SigmaS requires response regulator, Rrp2 (BB0763). Purified recombinant Rrp2 did not bind to the DNA upstream of B. burgdorferi rpoS in gel-mobility shift assays, nor did Rrp2-delta123, a truncated constitutively active form of Rrp2. Transcriptional activation of a rpoS-lacZ reporter gene in E. coli by Rrp2-delta123 was dependent on sigma54 but did not require DNA sequences upstream of the rpoS promoter. Similarly, quantitative RT-PCR experiments using a cat reporter gene in B. burgdorferi indicated that DNA sequences upstream of the sigma54-dependent rpoS promoter were not needed by Rrp2 to activate transcription of the rpoS P/O-cat reporter construct. Taken together, these findings suggest that unlike expression from most sigma54-dependent promoters, Rrp2 does not utilize an enhancer-like sequence to activate transcription of rpoS. Finally, quantitative RT-PCR showed that rpoN is required for expression of rpoS in B. burgdorferi as cultures enter stationary phase, and that hk2 is needed for optimal expression of rpoS during this time but is not absolutely essential. This suggests that, in addition to Hk2, another histidine kinase or small molecular weight molecule can serve as a phosphate donor to Rrp2, providing multiple signaling pathways for modulating the expression of RpoS and virulence related proteins, such as OspC. The focus of this aspect of our research is to further characterize this regulatory cascade involving SigmaS and Sigma54 in B. burgdorferi and to determine the extracellular signals that promote the survival of the bacterium in the tick midgut and in a human host.